We recently developed a bicameral (two compartments) cell culture system in which we can grow highly differentiated Sertoli cell monolayers at confluence. The cells rest on a permeable support (e.g., Millipore filter), impregnated with a reconstituted basement membrane such that the basal aspect of the cells can be bathed with a defined culture medium that is different from the medium at the apical surface of the cells. In this novel culture chamber we can sample and separately manipulate the fluids in the apical and basal sides of the epithelium. Thus, for the first time in culture, we are in an ideal position to mimic the two Sertoli cell compartments (basal and adluminal) that are present in vivo. Utilizing this new method of growing Sertoli cells, we propose to examine a number of polarized Sertoli cell functions in vitro that were not possible to investigate with conventional culture techniques. We plan to provide information on vectorial secretion by Sertoli cells of androgen binding protein, transferrin, and other proteins. The transport of substances across the Sertoli cell sheet from base to lumen, and in the opposite direction, will be examined. Purified populations of Leydig cells will be added to the basal part of the chamber, myoid cells will be grown on the under surface of the filter, and germ cells will be placed on top of the Sertoli cells in the apical compartment of the chamber. This organization of the cellular elements in culture is similar to the in vivo organization and should provide a useful model system to study cellular interactions and germ cell differentiation in vitro. Sertoli cells from isolated segments of seminiferous tubules (tubules at the same stage of the cycle) will be cultured in attempts to determine whether certain polarized functions of the Sertoli cells differ among the various stages. Finally, the dual compartment chamber system will be used to examine the role of the extracellular matrix in Sertoli cell differentiation and seminiferous tubule morphogenesis. These experiments should provide significant new information on the basic biology of the Sertoli cell, on the role of the Sertoli cell in germ cell differentiation, and on the interdependence of the cellular elements within the testis. We hope to be able to dissect factors which are responsible for specific steps of germ cell differentiation and this approach could very well have significance for the development of future male contraceptives.